The production of complex Asn (N)-linked oligosaccharides is highly regulated in mammalian development and is altered in specific human disease states. The structures play critical roles in ontogeny, as mouse embryos lacking a functional Mgat-1 allele were deficient in complex N- linked oligosaccharides and failed to develop further than day 9 of gestation. The objective of this proposal is to generate a viable animal model for loss-of function mutations in the alpha-mannosidase-II and Mgat-II genes, that both act immediately distal to Mgat-1 in the pathway of complex N-glycan synthesis. Such mutations are linked to Congenital Dyserythropoietic Anemia (CDA) type II in humans, likewise known as HEMPAS disease (hereditary erythroblastic multi-nuclearity with a positive acidified-serum lysis test). The HEMPAS phenotype includes aberrant complex N-linked oligosaccharide structures on leukocytes, platelets, in liver tissue and in the plasma membrane of multi-nucleated red blood cells. Some complex N-linked oligosaccharide structures therefore encode crucial functions in viable adults and prevent the emergence of specific disease states. Furthermore, it appears that the minimal complex N-glycan structure needed for mammalian viability with reproductive success has evolved to include the simple requirement for N-acetylglucosamine addition to the alpha-1-3 mannose of the high-mannose core, thereby forming the substrate for alpha-mannosidase-II, and subsequently, Mgat-II. However, this minimal structure appears to be insufficient for normal function since mutation is alpha-mannosidase-II or Mgat-II appears to generate recessive disease (HEMPAS) susceptibility in gametes. To test these hypotheses and to understand more fully the role of complex N-glycans in development and disease, we will generate in vivo mouse models of alpha-mannosidase-II and Mgat-II deficiency. This will entail (A) preparing gene-targeting vectors, isolating embryonic stem cells that have undergone gene-targeted homologous recombination at the alpha- mannosidase-II and Mgat-II loci, and producing chimeric mice that transmit such mutations in the germline. Such mice will enable the production of heterozygous and homozygous progeny that should mimic the human HEMPAS condition and provide material for (B) structure-function assessments of specific complex N-glycans in ontogeny and in post-natal function. With the expectation to gain a greater understanding of the HEMPAS phenotype, (C) a direct assessment of hemopoietic cell function, stem cell reconstitution potential and red blood cell physiology will be undertaken. Moreover, this proposal includes the use of a novel methodology for restricting gene ablation to specific cell lineages, as provided for by use of a Cre recombinase transgene and loxP-flanked DNA in the gene targeting constructs. This strategy overcomes experimental limitation that may occur should embryonic lethality occur in the mouse model. Importantly, this will permit (D) studies of alpha-mannosidase-II and Mgat-II function in specific cell lineages as they influence ontogeny and the multi-systemic nature of the HEMPAS phenotype.